At the present day, a half of the population in Japan suffers from cancer. However, if cancer is accurately diagnosed in the early stage and appropriately treated, better prognosis and QOL would be expected in many cases. Whether cells are cancer cells or not is determined as follows: cells (cytological diagnosis) or tissues (histological diagnosis) are taken from a suspected cancer site, observed by a cytotechnologist or a board certified cytopathologist for the presence of morphological anomalies, and diagnosed based on their experience and subjectivity. However, an early stage of cancer called a precancerous lesion or a borderline lesion called Class III may be difficult to be diagnosed when using the morphological approach alone. Indeed, these may result in disagreed interpretation among cytotechnologists or pathologists, or suspending of judgment in order to schedule a re-examination. When re-examined, patients may find that their cancer had continued to progress. Therefore, development of a method of evaluating cells which is more objective and definite than the present method is demanded for the sake of all of patients, pathologists, cytotechnologists, clinicians, and laboratory test providers.
Accurate diagnosis of borderline lesions is an earnest wish in clinical practice, and a serious issue which may change patients' future. Further, the conventional cytological diagnosis in which cells are morphologically observed one by one requires years of experience. Hence, training programs have been failing to provide necessary personnel in a timely fashion, resulting in increased workload on site and missed cases of cancer. These have been recognized as problems to be solved for years.
In a laboratory test for the identification of cancer, the less invasive “cytological diagnosis” (a laboratory test for the presence of cellular atypia including the N/C ratio, polarity, nuclear morphology and the like, and structural atypia in accumulation of cells and the like) is first performed to detect cancer cells solely based on morphology. When cancer cells or cells suspected to be cancerous are detected, histological diagnosis which is capable of providing a more detailed identification is performed, and the results therefrom are used to decide on a treatment strategy.
Further, “cytological diagnosis” is frequently used for the purposes of: conveniently examining the presence of cancer at medical checkup and the like; examining the presence of dissemination over peritoneum, pleura, and the like by withdrawing a coelomic fluid such as an ascitic fluid or a pleural fluid during a cancer operation or for diagnosis; or detecting the presence of cancer such as tumors in the urinary system by testing urine. In cases where histological diagnosis cannot be performed, such as when an ascitic fluid, a pleural fluid and the like are tested, or when the presence of renal pelvis cancer is investigated, and the like, cytological diagnosis needs to be performed to decide on a treatment strategy.
In Japan, except for in some advanced institutions, cytological diagnosis is commonly performed as follows: a sample taken from a patient is smeared onto a glass slide, fixed with alcohol, and then stained and embedded. Cell morphology is then observed under a microscope. Cytological diagnosis can be performed quickly and conveniently, but cytotechnologists dedicated for the tests are required to examine one by one a large number of cells contained in every glass slide for morphological and nuclear anomalies repeatedly every day. Years of experience and skills are required to find cancer cells quickly and reliably without missing any.
Moreover, if cancer cells, no matter how few they are, are found within the accumulated cell populations, the test concludes that the sample has cancer. Since overlooking of cancer cells may lead to a delay in accurate diagnosis, concentration, patience, and skills for finding cancer cells without missing any are required. However, it cannot be denied that such abilities of cytotechnologists may vary between individuals.
In addition, for cells suspended in an ascitic fluid or urine, target cells for observation are present in a state where they are detached from the original tissues. Such cells are away from supplies of blood flows and nutrition, becoming more susceptible to cell degeneration. Therefore, it is not uncommon that these cells undergo morphological changes to have densely stained nuclei, increased. N/C ratios (nucleus/cytoplasm ratios), or the like, resulting in difficulty in distinguishing them from cancer cells. These cells may be classified as so-called Class III for which benign-malignant discrimination is difficult, hindering decision making on the treatment strategy, resulting in watch-and-wait or re-examination. Further, the range of classification of Class III may vary depending on the experience and subjectivity of individual cytotechnologists and board certified cytopathologists. This may delay the start of treatment to the detriment of patients. However, the current cytological diagnosis relying solely on morphological information is obviously less than satisfactory for solving the aforementioned problems.
Moreover, in some cases, observation under a light microscope cannot provide a clear view of target cells due to the presence of a large amount of blood cells, hindering accurate diagnosis. In order to improve this situation, the liquid-based cytological diagnosis (LBC) has been proposed as a method of preparing a sample. Although LBC is useful for reducing human factors in preparing specimens, there is a report stating that it fails to produce significant difference in benign-malignant discrimination over the conventional method.
Whether cells are cancer cells or not is conventionally determined by cytotechnologists and board certified cytopathologists based on the following morphological characteristics of cells, among others:
1) an increased ratio of nucleus to cytoplasm (the N/C ratio);
2) anomalies in cell morphology, including the position of nucleus in a cell and the morphology of nucleus chromatin;
3) anomalies in the clustering conditions of cells (structural atypia).
However, such diagnosis relying on morphological anomalies is made based on the subjectivity of cytotechnologists and board certified cytopathologists. Therefore, cells may not be diagnosed when they are at the borderline between benign and malignant, or may not be able to be diagnosed solely based on cell morphology. As described above, diagnosis based on subjectivity may provide different results depending on opinions of observers, or may result in inconclusive results. As one example, there is 10% or more Class III classification in tests for recurrence of bladder cancer. This is a serious problem that needs solving.
Logically speaking, although specimens classified as Class III would be either cancerous or non-cancerous, the distinction between “cells being suspicious for malignancy” and “cells not being suspicious for malignancy” is not discrete. As such, in order to reduce Class III, there are demands for an objective measure which can be used in place of the current cytological diagnosis which has a subjective aspect as described above, or well-defined indexes which can allow for determination without relying on amount of experience.
Further, in view of the current situation where anomalies are visually searched by skilled cytotechnologists one by one when a large number of laboratory tests for cytological diagnosis requiring immediate attention are piled up every day, a guideline for reliably finding anomalies is required in order to avoid false negatives due to overlooking, of cancer.
Immunocytochemical staining may be performed, when determination by common staining such as Papanicolaou stain, Giemsa stain and the like, which are used as the conventional method of cytological diagnosis of cancer, needs to be supplemented. However, no single antibody can provide benign-malignant discrimination on its own, and thus multiple antibodies are required to be combined. Even in that case, decisive benign-malignant discrimination will be difficult.
In addition, it is noted that the current cytological diagnosis is solely based on “morphologically detectable anomalies” of dead cells fixed by alcohol, and is not a method for observing “functional anomalies of cells” which may not necessarily be manifested as morphological changes. Common cytological diagnosis alone cannot determine whether or not a cell in a specimen is functionally normal even if it looks like normal cell morphology. For example, a cell, which is morphologically indistinguishable from a normal cell, may already be infected with a virus having a high risk of subsequent cancerization, or may already show an abnormal cell function. If that is the case, definitive diagnosis cannot be obtained until morphological anomalies are clearly observed. This may delay the start of treatment due to repeated tests, resulting in a detriment of patients.
The present inventors have developed 2-[N-(7-nitrobenz-2-oxa-1,3-diazole-4-yl)amino]-2-deoxy-L-glucose (2-NBDLG), a green fluorophore NBD-labeled, novel derivative of non-naturally occurring L-glucose, which does not bind to a glucose transporter GLUT. Further, the present inventors have found that 2-NBDLG visualizes cancer cells due to its specific uptake into them, when administered to in vitro culture cells such as pancreatic cancer cells or to cancer-bearing mice, allowing for imaging of a cancer cell cluster consisting of cancer cells with various cell states (Patent Document 1).
The present inventors also have developed 2-amino-2-deoxy-L-glucose (2-TRLG), a plasma membrane-impermeable fluorescent L-glucose derivative bearing a red fluorophore sulforhodamine 101 being attached at position 2 via a sulfonamide bond. Further, in experiments in vitro where 2-TRLG and 2-NBDLG are applied to cultured cells, the present inventors have found that both 2-NBDLG and 2-TRLG are taken up into cells having damaged plasma membranes as well as cells showing nonspecific uptake such as phagocytosis, allowing for discrimination of cells which have taken up 2-NBDLG only (Patent Document 1).    Patent Document 1: PCT International Publication No. WO2012/133686    Non Patent Document 1: Yamamoto et al., Bioorg. Med. Chem. Lett. 21:4088-4096, 2011